Antibiotic MM 13902

ABSTRACT

It has been found that a novel antibiotic can be obtained by the fermentation of certain strains of Streptomyces olivaceus and related organisms. In addition to being a potent antibiotic this new material which we have designated MM 13902 acts synergistically with penicillins and cephalosporins.

CROSS REFERENCE

This application is a continuation-in-part application of Ser. No.716,668 filed Aug. 23, 1976, now abandoned which is a divisionalapplication of Ser. No. 559,803 filed Mar. 19, 1975, now abandoned.

BACKGROUND TO THE INVENTION

British Pat. No. 1,363,075 disclosed that a useful β-lactamase inhibitorcould be obtained by the fermentation of certain strains of Streptomycesolivaceus. Until our present invention, it was believed that thematerial disclosed in British Pat. No. 1,363,075 was substantially pure.However, we have discovered that this is not so and that a minorcomponent of that material can be isolated and has potent antibacterialactivity. This new material is designated MM 13902 and it is nowbelieved to be responsible for a part of any antibacterial activitypresent in the material of British Pat. No. 1,363,075 although it isresponsible for only a very minor part of β-lactamase inhibitoryactivity exhibited by that material. Naturally, nothing in thisspecification should be construed as claiming material as disclosed inBritish Pat. No. 1,363,075. Other β-lactamase inhibitors are known to beproduced by strains of Streptomyces, for example those disclosed inGerman Published Patent Application No. 2,340,005, but such knownmaterials have not been demonstrated as having the potent antibacterialactivity of the kind possessed by MM 13902. U.S. Pat. No. 3,950,357disclosed that Streptomyces cattleya produced the antibiotic Thienamycinwhich has the structure: ##STR1## Based on the present knowledge of thestructure of MM 13902, significant structural differences are apparentbetween thienamycin and MM 13902.

DESCRIPTION OF THE INVENTION

The present invention provides the antibacterial agent designated hereinMM 13902 and its salts.

MM 13902 is a solid carboxylic acid which in the form of a substantiallypure sodium salt has the following characteristics:

i. It is highly soluble in water, soluble in methanol and substantiallyinsoluble in chloroform, diethylether and hydrocarbons.

ii. In aqueous solution, it has a characteristic ultra-violet spectrumwith absorption maxima one of which is at about 305 nm.

iii. When present at 0.4% w/w in a freshly prepared KBr disc, it has acharacteristic infra-red spectrum which has absorption maxima at, interalia, about 3450, 2950, 1750, 1620, 1510, 1400 and 1260 cm⁻¹.

iv. It has a characteristic n.m.r. spectrum when taken in D₂ O whichspectrum possesses, inter alia, (a) a pair of low field doublets centredat approximately 2.85ρ and 4.00ρ with coupling constants ofapproximately 14 Hz; (b) a doublet centred at approximately 8.55ρ and(c) a sharp singlet at approximately 8.00ρ.

v. It possessed antibacterial activity against various speciesincluding, inter alia, strains of Staphylococcus aureus, Bacillussubtilis, Escherichia coli, Klebsiella aerogenes, Proteus mirabilis,Salmonella typhi and Pseudomonas aeruginosa.

vi. When mixed with ampicillin it synergizes its activity againstorganisms including strains of Escherichia coli, Klebsiella aerogenes,Proteus mirabilis, Proteus morganii and Staphylococcus aureus Russell.

The pure di-sodium salt of MM 13902 has an I₅₀ (as hereinafterdescribed) of between 0.001 μg/ml and 0.0001 μg/ml against theβ-lactamase of Escherichia coli B 11.

When run on cellulose in a thin layer chromatography system the puredi-sodium salt of MM 13902 has the following approximate R_(f) values:

(a) n-butanol/isopropanol/water--7:7:6 v/v; R_(f) =0.85

(b) isopropanol/water--7:3 v/v; R_(f) =0.70

(c) n-butanol/ethanol/water--7:7:6; R_(f) =0.79

(d) n-propanol/water--4:1; R_(f) =0.68

From another viewpoint, MM 13902 may be characterized as a sulphurcontaining antibacterial agent, salts of which are produced during thecultivation of Streptomyces olivaceus ATCC 31126 and in the form of anaqueous solution of its di-sodium salt has UV absorption maxima at about305 nm and at about 225 nm substantially as shown in FIG. 1 herein.

Alternatively, MM 13902 may be characterized as an antibacterial agentwhich in the form of its substantially pure di-sodium salt has aninfra-red absorption spectrum substantially as shown in FIG. 2 whentaken in a freshly prepared 0.4% w/w KBr disc and which in the form ofits substantially pure di-sodium salt has n.m.r. spectrum substantiallyas shown in FIG. 3 when taken in a freshly prepared solution in D₂ O.

The material MM 13902 is of the molecular formula C₁₃ H₁₆ O₈ N₂ S₂.

The material MM 13902 is believed to have the structural formula:##STR2##

The material MM 13902 tends to be unstable when in the unsalted acidform. Accordingly a preferred aspect of this invention provides salts ofMM 13902. Suitably the salts of MM 13902 are di-basic salts. Mostsuitably these salts are those formed with pharmaceutically acceptableions such as sodium, potassium, calcium, magnesium, aluminium,conventional substituted ammonium ions and the like. Particularlysuitable salts are alkali metal salts such as the sodium or potassiumsalts, for example, the di-sodium or di-potassium salts.

Suitably the MM 13902 and its salts as provided by this invention are atleast 50% pure, more suitably at least 70% pure and preferably 80% pure,for example 90-100% pure.

In a further aspect this provides a pharmaceutical composition whichcomprises MM 13902 as hereinbefore described or a salt thereof togetherwith a pharmaceutically acceptable carrier.

Most suitably the compositions of this invention contain a sodium orpotassium salt of MM 13902, for example, the di-sodium or di-potassiumsalt of MM 13902.

Such compositions may be in a form suitable for oral, topical orparenteral use. For example, tablets, capsules, creams, syrups,reconstitutable powders and sterile forms suitable for injection orinfusion may be used. Such compositions may contain conventionalpharmaceutically acceptable materials such as diluents, binders,colours, flavours, preservatives, disintegrants and the like inaccordance with conventional pharmaceutical practice in a manner wellknown to those skilled in the formulation of antibiotics such aspenicillins and cephalosporins.

The MM 13902 or its salts may be present in the composition of theinvention as sole therapeutic agent or it may be present together with aβ-lactam antibiotic. Suitable β-lactam antibiotics include those knownto be susceptible to β-lactamases and also having some intrinsicresistance to β-lactamases. Such β-lactam antibiotics includeampicillin, amoxycillin, benzylpenicillin, phenoxymethylpenicillin,propicillin, cephaloridine, cefoxitin, cephalothin, cephalexin,carbenicillin, ticarcillin and in vivo hydrolysable esters of suchcompounds such as the phenyl, tolyl or indanyl esters of carbenicillinor ticarcillin or the acetoxymethyl, pivaloyloxymethyl or phthalidylesters of ampicillin, benzylpenicillin, amoxycillin, cephaloridine,cephaloglycin and the like.

The ratio of MM 13902 or salt thereof to β-lactam antibiotic is normallybetween 10:1 and 1:10, for example, between 3:1 and 1:3.

The total quantity of antibacterial agents present in any unit dosageform will normally be between 50 and 1500 mgs and will usually bebetween 100 and 1000 mgs.

Preferred unit dosage compositions according to this invention may beadministered one or more times a day, for example, 2 to 4 times a day,in the treatment of dieases of the urinary tract, respiratory tract,soft tissues and the like. Thus the compositions may be used in thetreatment of such diseases as bronchitis, gonorrhea, otitus media,mastitis and the like.

In one of its aspects, this invention provides a process for thepreparation of MM 13902 or a salt thereof which compriseschromatographically separating a solution of MM 4550 (Complex) ashereinafter described into fractions consisting essentially of asolution of MM 13902 or a salt thereof and other fractions and isolatingthe MM 13902 or a salt thereof from solution.

By those fractions "consisting essentially of a solution of MM 13902 ora salt thereof" is meant that either the only antibiotic materialpresent in that solution is MM 13902 or a salt thereof or that if anyother antibiotic material is present then it is there to a lesser extentthan the MM 13902 or salt thereof. More suitably MM 13902 or a saltthereof represents 70%, most suitably 80% and preferably 90-100%, of theantibiotic material present in the fraction. We have found that anacceptable method of determining which fractions consist essentially ofa solution of MM 13902 or a salt thereof is to moniter the uv absorptionspectrum of each sample. Those fractions showed a uv spectrum similar tothat of FIG. 1 normally contain MM 13902 or a salt thereof substantiallyfree from other antibiotic material such as MM 4550A as hereinafterdescribed. In general those fractions showing a distinct UV absorptionmaximum at about 305 nm are of the desired purity.

Normally the preceeding process is used for the isolation of MM 13902 asa di-basic salt. If a monobasic salt of MM 13902 or the free acid MM13902 per se is required, they may be prepared by the acidification of adi-basic salt of MM 13902 because in general the monobasic salts of MM13902 and the free acid MM 13902 are not sufficiently stable to isolatefrom mixtures such as MM 4550 (Complex) as hereinafter described. Thesemonobasic salts and free acid are not readily obtainable owing to theirlow stability.

As previously stated, we believe that the chromatographic isolation ofMM 13902 is best carried out using a salt of MM 13902 such as thedi-sodium salt. Salts of MM 13902 are more soluble in aqueous solventsystems than in highly lipophilic solvents and consequently it ispreferred to use aqueous solvent systems in the chromatographicpurifications used in this invention.

In our hands aqueous solutions of electrolytes buffered to approximatelyneutrality have proved suitable for use in conjunction with polarsupport materials such as basic ion-exchange resins. Thus an aqueoussolution of sodium chloride buffered to about pH 7 with a conventionalbuffer such as a phosphate buffer may be used in conjunction withsupport materials which may contain tertiary amino groups or quaternaryammonium groups. We have found that basic ion-exchange celluloses andbasic ion-exchange cross linked dextrans are suitable support materialsand that QAE Sephadex A25 in particular is a highly suitable supportmaterial especially when the solvent system comprises 0.7 m sodiumchloride in pH 7 phosphate buffer. (`Sephadex` is a Registered TradeMark).

Alternatively, in our hands solvent systems comprising mixtures of waterand water viscible organic solvent such as a lower alkanol have provedsuitable for use in conjunction with inert support materials such assilica gel or cellulose. A particularly suitable solvent system for usewith cellulose is a mixture of water and isopropanol, especially a7:3-isopropanol:water mixture.

However, the product of the above procedures frequently contains a veryhigh proportion of sodium chloride so that it is beneficial to de-saltthe pooled solutions. De-salting may be effected by passing the solutiondown a column containing a lipophilic material onto which the MM 13902is adsorbed but which does not adsorb the sodium chloride. Suitablecolumn materials include polystyrenes such as Amberlite XAD 4 and gelfiltration agents such as cross linked dextrons such as Sephadex G 25and polyacrylamide gels such as Biogel P2 (`Amberlite`, `Sephadex` and`Biogel` are Trade Marks). The antibiotic may be eluted from the columnusing water, aqueous methanol or the like.

When the desired solutions are obtained by the above process thedi-basic salt of Mm 13902 may be obtained in solid form by the removalof the solvent under mild conditions. We have found that an acceptablemethod of obtaining such solid material is to freeze-dry the pooledfractions containing MM 13902.

If desired the preceeding process may be carried out in two or moresteps. For example, a solution of MM 4550 (Complex) may be separatedinto fractions comprising MM 13902 or salt thereof contaminated with upto its own weight of other antibacterial agents, the fractions may befreeze dried to yield a material containing, for example, about 50-60%of MM 13902 or a salt thereof and this material may then berechromatographed to yield a material which contains, for example,90-100% of MM 13902 or a salt thereof.

For the purpose of this specification the term "MM 4550 (Complex)" isused to describe the material originally designated as MM 4550 inBritish Pat. No. 1,363,075. MM 4550 (Complex) as produced on repetitionof the Examples of British Pat. No. 1,363,075 is an impure materialwhich contains in various proportions salts of MM 4550A (as describedhereinafter) and salts of MM 13902 and considerable quantities of othermaterials. MM 4550 (Complex) does not have the characteristicultra-violet spectrum of MM 13902. The I₅₀ (as described hereinafter) ofMM 4550 (Complex) produced by repetition of the Examples of British Pat.No. 1,363,075 is not normally below 0.0004 μg/ml. The ratio of salts ofMM 4550A and salts of MM 13902 present in MM 4550 (Complex) is believedto be highly variable and is thought to depend on such factors as thestrain of organism used and/or the exact isolation techniques used butit has been generally found that the complex contains more MM 4550A thanMM 13902. The preparation of MM 4550 (Complex) is described hereinafterin the section relating to "Descriptions".

For the purpose of this specification the term "MM 4550A" is used todescribe a substantially pure compound which is a potent β-lactamaseinhibitor and which also possesses a certain degree of antibacterialactivity. The properties of MM 4550A are described hereinafter in thesection relating to "Descriptions".

In an alternative view, this invention provides a process for thepreparation of MM 13902 and its salts which process comprises thecultivation of a MM 13902 producing strain of Streptomyces andthereafter recovering the MM 13902 or salt thereof from the culture.

For this process we have found that MM 13902 producing strains arestrains of Streptomyces olivaceus and related organisms as describedhereinafter.

Most suitably the organism used is a strain of Streptomyces olivaceussuch as ATCC 21379, 21380, 21381, 21382, 31126 or mutants thereof.

A particularly preferred organism for use in this process isStreptomyces olivaceus ATCC 31126.

When used herein, the term "cultivation" means the deliberate aerobicgrowth of an organism in the presence of assimilable sources of carbon,nitrogen, sulphur and mineral salts. Such aerobic growth may take placein a solid or semi-solid nutritive medium, or in a liquid medium inwhich the nutrients are dissolved or suspended. The cultivation may takeplace on an aerobic surface or by submerged culture. The nutritivemedium may be composed of complex nutrients or may be chemicallydefined. In our hands we have found media containing complex nutrientssuch as yeast extract, soya bean flour and the like to be particularlysuitable. We have also found that the addition of cobalt ions, sulphateions and calcium carbonate to be beneficial.

We have found that cultivation at a temperature of 28°±2° C. givesacceptable yields of antibiotic and that a good time to harvest thebroth is 2-3 days after the initiation of fermentation.

When used herein the term "mutant" includes any mutant strain whicharises spontaneously or through the effect of an external agent whetherthat agent is applied deliberately or otherwise. Suitable methods ofproducing mutant strains include those outlined by H. I. Adler inTechniques for the Development of Micro-organisms in "Radiation andRadioisotopes for Industrial Micro-organisms", Proceedings of aSymposium, Vienna, 1973, p. 241, International Atomic Energy Authorityand these include:

i. Ionising radiation (such as X- and Y- rays), uv light, uv light plusa photosensitizing agent (such as 8-methoxypsoralen), nitrous acid,hydroxylamine, pyrimidine base analogues (such as 5-bromouracil),acridines, alkylating agents (such as mustard gas, ethyl-methanesulphonate), hydrogen peroxide, phenols, formaldehyde, heat, and

ii. Genetic techniques such as recombination, transformation,transduction, lysogenisation, lysogenic conversion and selectivetechniques for spontaneous mutants.

We have found that use of a mutation promoting agent can lead to theproduction of organisms which have the ability to produce enhancedquantities of the desired antibiotics. For example, irradiation ofcultures of Streptomyces olivaceus ATCC 21379 followed by isolation ofthe resulting strain which appeared to produce the largest zone ofactivity on the KAG assay as hereinafter described lead to the isolationof Streptomyces olivaceus ATCC 31126 which is a preferred strain for usein this invention.

In general, all isolation and purification procedures used in obtainingthe desired antibiotic should take place at non-elevated temperatures,for example, below 20° C. and more suitably not above 12° C.

The desired product is normally obtained predominantly from the culturefiltrate so that the preferred initial step in the isolation process isthe removal of solid material from the fermentation, for example byfiltration.

An impure preparation of MM 13902 or a salt thereof may be obtained fromthe clarified culture filtrate by absorbing the MM 13902 or a saltthereof onto an active material such as active carbon or the like andthereafter eluting the desired substance from the active material usinga solvent such as aqueous acetone. Normally this process is carried outon a di-basic salt of MM 13902.

Alternatively an impure preparation of MM 13902 or a salt thereof may beobtained from the culture filtrate by extraction using a lipophilicammonium salt and a water immiscible solvent. This is frequently moreeffective than the process preceeding absorbtion described above. (TheMM 13902 may be obtained as the substituted ammonium salt by evaporationof the organic solvent under reduced pressure.) Preferably the initialsolution of the MM 13902 substituted ammonium salt is then backextracted into an aqueous phase by using a solution of an alkali metaliodide such as sodium iodide. This last process variant generally leadsto preparation of an aqueous solution of an impure di-basic salt of MM13902.

The impure forms of MM 13902 or its salts as described above arenormally subjected to the chromatographic procedures hereinbeforedescribed in order to produce material of acceptable purity.

The following Descriptions elucidate general information useful in theisolation of antibacterial compounds. The following Examples areillustrative of aspects of the invention.

DESCRIPTION 1 I₅₀ Determination

The I₅₀ value is the amount of material required to give 50% inhibitionof hydrolysis of ampicillin by the β-lactamase enzyme of Escherichiacoli B11, an organism containing an R factor controlled β-lactamase.This β-lactamase is classified as a type IIIa enzyme according to theclassification of Richmond and Sykes [Adv. in Microbiol. Physiol. 9, 31(1973)]

The rate of β-lactamase hydrolysis of ampicillin to its penicilloic acidcan be followed by a starch-iodometric assay in which one measures therate of formation of penicilloic acid by following the decolorization ofa starch-iodine complex. This method and the preparation of theβ-lactamase are described in a paper by Cole M., Elson S., and FullbrookP.D. (Biochemical Journal 1972, 127, 295-308). A slight modification ofthe above method was used in that the sample of inhibitor waspreincubated with the enzyme for 15 minutes at 37° C. prior to addingthe substrate ampicillin. The procedure was as follows:

    ______________________________________                                        Reagents                                                                      ______________________________________                                        Buffer:  0.05M pH7 potassium phosphate buffer                                 Starch/iodine                                                                          Prepared as described by Novick, Biochemical J.                      solution:                                                                              (1962) 83, 236                                                       Substrate:                                                                             Ampicillin 40 μg/ml in buffer                                     β-lactamase                                                                       Prepared from E. coli B11 as described by Cole                       Enzyme:  et al, Biochem. J. (1972) 127, 295. The dilution                              of the enzyme preparation in buffer was such as                               to give a fall of about 0.3 optical density                                   units per 100 secs. in the uninhibited reaction.                              Other β-lactamase producing strains of E. coli                           may be used, in particular those carrying an R                                factor for example R TEM.                                            ______________________________________                                    

Conditions--

The reactions were carried out in 1 cm cuvettes at 37° C. in an SP 800Pye Unicam spectrophotometer. This instrument can carry four samplecuvettes and their corresponding blanks. The first cuvette was used forthe control reaction and contained no inhibitor. The 2nd, 3rd and 4thcuvettes were used for various dilutions of the inhibitor. Thus:

    ______________________________________                                                             Sample    Blank                                          Reagents             Cuvette   Cuvette                                        ______________________________________                                        Starch/iodine reagent                                                                              1.0 ml    1.0 ml                                         E. coli β-lactamase                                                                           0.1 ml    --                                             Buffer               0.3 ml    0.4 ml                                         Inhibitor of buffer  0.1 ml    0.1 ml                                         Substrate (added after incubation                                                                  1.0 ml    1.0 ml                                         of above mixtures for 15 mins.                                                at 37° C.)                                                             ______________________________________                                    

The reactions were followed by recording optical density change at 590nm and measuring the velocity of the reaction as optical density changeper 100 secs. during the 3-6 minute time interval. The inhibitor samplewas diluted until a dilution was reached which gave 50% of the rate ofreaction seen in the no-inhibitor control.

DESCRIPTION 2 The KAG Assay

The KAG assay is a method for determining the presence of a β-lactamaseinhibitor in fermentation broths or during stages in the isolation.Molten nutrient agar at 45° C. is seeded with a suitable β-lactamaseproducing strain of Klebsiella aerogenes and then mixed with asufficient quantity of a sterile solution of penicillin G to give aconcentration of 6 μg/ml of penicillin G in the agar. The agar is thenpoured into a petri dish and after solidification equally spacedcylindrical wells are made in the layer of agar by using a sterile metalcutter. The solutions to be tested are introduced into the wells. Thedish is then incubated at a constant temperature between 27° C. and 37°C. During the period of incubation, any antibiotic in the test solutiondiffuses out from the well into the agar and there inhibits the actionof β-lactamase produced by the Klebsiella cells. The penicillin G isthus protected from destruction by β-lactamase and is present insufficient concentration to prevent the growth of the Klebsiella. Inthose parts of the agar to which the antibiotic has not diffused insufficient concentration, the penicillin G is destroyed by theβ-lactamase, allowing dense growth of the Klebsiella to develop. Clearcircular zones of inhibition of Klebsiella growth are thus formed aroundthe wells containing antibiotic the size of each zone depending on theconcentration of antibiotic in the solution under test. The potency(arbitrary units/ml) of test solutions is obtained by reference to astandard line of log.conc. antibiotic v. zone diameter. This assaysystem may also be used for bioautography.

DESCRIPTION 3 Preparation of MM 4550 (Complex) comparable to thatdisclosed in British Pat. No. 1,363,075

Streptomyces olivaceus ATCC 21379, was grown for 7 days at 28° C. on asolid agar slant in a Roux bottle. The agar medium had the followingcomposition:

    ______________________________________                                        Constituent           Amount (g/l)                                            ______________________________________                                        Yeast Extract             10.0                                                Glucose Monohydrate       10.0                                                Agar                      15.0                                                Tap Water        to       1 l                                                 ______________________________________                                         [The "Yeast extract" was "Yeatex" as supplied by Bovril Food Ingredients,     P.O. Box 18, Wellington Road, Burtonon-Trent, U.K., and the "Agar" was        supplied by Oxoid Limited, Southwark Bridge Road, London, S.E.1., U.K.   

The medium was adjusted to pH 6.8 before sterilization. 50 ml of steriledeionised water containing 0.02% Tween 80 [Registered Trade Mark; Tween80 is a polyoxyethylene sorbitan mono-oleate] was added to a Roux bottleculture and the spores suspended by shaking. This spore suspension wasthen added as inoculum to 75 l of sterilized seed stage medium in a 100l stainless steel fermenter. The composition of the seed stage mediumwas as follows:

    ______________________________________                                        Constituent           Amount (g/l)                                            ______________________________________                                        Soya-bean Flour           10.0                                                Glucose Monohydrate       20.0                                                Tap Water        to       1 l                                                 ______________________________________                                         [The "Soyabean Flour" was Arkasoy 50 as supplied by the British Arkady Co     Ltd., Old Trafford, Manchester].                                         

To control foaming 50 ml of 10% v/v Pluronic L81 (Registered Trade Mark)in soya-bean oil was added to the fermentation medium beforesterilization. [Pluronic L81 was supplied by Jacobsen van den Berg U.K.Ltd., 231 The Vale, London, W.3., U.K., and is a block polymer ofethylene oxide and propylene oxide].

The medium was steam sterilized in the fermenter for 20 mins at 120° C.The seed stage culture was stirred at 340 r.p.m. with a 8.5 inchdiameter vaned disc agitator and supplied with 150 l/min sterile airthrough an open ended sparger. The culture vessel was fitted withbaffles. The temperature was controlled at 28° C. and after incubationunder these conditions for 45 hours, 7.5 l of this seed culture wasadded as inoculum to 150 l sterile fermentation medium in a 300 lstainless steel fermenter. The fermentation medium had the followingcomposition:

    ______________________________________                                        Constituent             Amount (g/l)                                          ______________________________________                                        Soya-bean Flour (Arkasoy 50)                                                                              10.0                                              Glucose Monohydrate         20.0                                              Chalk (Precipitated Calcium                                                    Carbonate)                  0.2                                              Cobalt Chloride (CoCl.sub.2, 6H.sub.2 O)                                                                    0.001                                           Tap Water           to      1 l                                               ______________________________________                                         300 ml of 10% Pluronic L81 in soya-bean oil was added to prevent foaming.     The fermentation was harvested after 48 hours and clarified by     centrifugation. The clarified brew gave 50% inhibition in the enzyme assay     at a dilution of 1 in 100,000. 100 l of the clarified brew was stirred     with 12 kg. wet weight of Whatman DE32 (Registered Trade Mark) ion     exchange cellulose in the acetate form, [as aupplied by H. Reeve Angel &     Co., 14 New Bridge Street, London E.C.4, U.K; the material is a     microcrystalline cellulose substituted by diethylaminoethyl groups].

The slurry was filtered and the MM 4550 (Complex) was eluted from thecellulose with 12 l of 0.5 M potassium sulphate. The extract wasconcentrated to 6 l in a climbing film evaporator under vacuum and below30° C. Much of the potassium sulphate was precipitated by the additionof 12 l of acetone. The solution was filtered and concentrated to 200 mlby evaporation under vacuum below 30° C. The concentrate was loaded ontoa 76 mm×2 m column of Amberlite XAD-4 resin (Registered Trade Mark) [asmanufactured by Rhom & Hass Co., Philadelphia, U.S.A.; the material is anon-ionic polystyrene resin], eluted with deionised water and theelutate was collected in 140 ml. fractions. Active fractions, asdetected by the agar diffusion assay were bulked (2.2 l) andconcentrated to 275 ml by ultrafiltration using an Amicon UM-05 membrane(150 mm diameter) (Registered Trade Mark) [as supplied by Amicon Ltd.,57 Queen's Road, High Wycombe] under nitrogen pressure of 60 p.s.i. Theconcentrate was freeze dried to yield 2.2 g of brown powder (I₅₀ 0.004μg/ml). 1 g of the powder was dissolved in 1 l of 0.2 M sodium sulphateand mixed with 1 l of 2% w/v tetra-n-butylammonium hydrogen sulphate (assupplied by AB Astra, Sodertalje, Sweden) in dichloromethane. Thedichloromethane phase was separated by gravity, cooled to -70° C.,filtered to remove ice and concentrated by evaporation to 20 ml. 400 mlof 40°-60° C. petroleum spirit was added to the concentrate and theprecipitate collected by centrifugation. The precipitate was redissolvedin dichloromethane (10 ml) and extracted with water (10 ml) containingbarium iodide (80 mg) and barium carbonate (70 mg). The phases wereseparated, the aqueous phase filtered and adjusted to pH 6.5 Thesolution was freeze dried to yield a yellow powder. The solid was washedwith acetone to dissolve out excess barium iodide and the pale yellowsolid recovered by centrifugation and dried in vacuo. The yield was 13mg. (I₅₀ 0.0004 μg/ml).

DESCRIPTION 4 Demonstration of Carbon Adsorption of Culture Filtrateuseful in obtaining material as described in British Pat. No. 1,361,075

Culture filtrate obtained as in Description 3 gave a zone diameter of 17mm in a hole-in-plate agar diffusion antibacterial assay againstKlebsiella aerogenes; a zone diameter of 38 mm in the KAG assay and I₅₀at a dilution of 1 in 150,000. The clarified culture filtrate (170 l) at5° C. was percolated by upward flow through a 9" diameter column packedto a height of 16" with active charcoal (Farnell BO, as supplied byDearborn Chemicals Ltd., Dilton, Widnes, Lancs., 60-80 mesh pre-washedwith N HCl and buffered at pH 6 with phosphate) at a flow rate of800-1000 ml/min. Brew was washed from the carbon with deionised water(10 l) and eluted with 20% acetone at 20° C. The active fractionsamounting to 10 l were concentrated by evaporation in vacuo, below 30°C. to 6 l and freeze dried to yield 282 g of crude MM 4550 (Complex)preparation giving an I₅₀ of 0.05 μg/ml. The recovery of enzymeinhibitory activity was 22%.

An alternative activated charcoal which gives similar results isDarcogranular carbon (as supplied by Honeywill-Atlas Ltd., Mill Lane,Carshalton, Surrey).

DESCRIPTION 5 Demonstration of Ion Exchange Resin Chromatography ofCulture Filtrate useful in obtaining material as described in BritishPat. No. 1,363,075

A 1 cm internal diameter column was packed to a height of 6 cm (bedvolume 4.7 ml) with Dowex 21 K ion-exchange resin (20-50 U.S. mesh,chloride form) (Dowex 21 K was supplied by B.D.H.Chemicald Ltd., Poole,Dorset, U.K. and is a polystyrene-divenylbenzene resin containing basicgroups). The bed of resin was then washed with approximately 4×4.7 ml of5% aqueous methanol, followed by 1×4.7 ml of distilled water. Two litersof culture filtrate was obtained essentially as described in Description3. The resin was then washed with 50% aqueous methanol to removeimpurities.

The MM 4550 (Complex) was eluted from the resin with 5% NaCl in 50%aqueous methanol. Table 1 below shows the results obtained in terms ofunits of activity. The MM 4550 (Complex) content of culture filtrate wasarbitrarily set at 8 units/ml. The eluted fractions from the column wereassayed using an antibacterial diffusion method against Klebsiellaaerogenes and the activity units were calculated by reference to astandard line prepared by plotting zone diameter against units/ml forvarious dilutions of culture filtrate (i.e. the culture filtrate wasused as a standard).

It can be seen that the column was an effective way of concentrating theMM 4550 (Complex). Fractions 2-5 inclusive contained 60% of the activityin a total volume of only 37 ml. The total dry weight of these fractionswas about 210 mg whereas 35 g of solids had been added to the column.

                  TABLE 1                                                         ______________________________________                                        Results Of Chromatography On Dowex 21K                                                          Volume                                                      Sample    pH      (ml)      Units/ml                                                                              Total units                               ______________________________________                                        Culture filtrate                                                                        6.5     1970      8       15760                                     Percolate 1                                                                             6.9     550       <1      <100                                      Percolate 2                                                                             7.0     525       <1      <100                                      Percolate 3                                                                             7.0     595       <1      <100                                      Percolate 4                                                                             7.0     300       <1      <100                                      Eluate 1  7.8     7.0       84      588                                       Eluate 2  7.8     7.0       328     2296                                      Eluate 3  7.7     8.5       440     3740                                      Eluate 4  7.7     10.0      320     2200                                      Eluate 5  7.6     11.5      160     1840                                      Eluate 6  7.5     11.0      80      880                                       Eluate 7  7.6     14.2      66      937                                       Eluate 8  7.6     20.0      33      660                                                                   Total:  13141                                     ______________________________________                                    

DESCRIPTION 6 Demonstration of Ion-Pair Extraction of Culture Filtrateuseful in obtaining material as described in British Pat. No. 1,363,075

Sodium sulphate (248 g) was added to clarified culture filtrate (10 l)prepared as described in Description 3 and the solution extracted with2% tetra n-butyl-ammonium hydrogen sulphate in dichloromethane (10 l) bystirring for 30 minutes. The phases were separated and thedichloromethane phase cooled to 2° C. A small quantity of suspendedwater was removed by filtration through Whatman No. 1 PS paper. Thedichloromethane solution was concentrated to 20 ml by evaporation invacuo below 30° C. The addition of 400 ml of petroleum ether (40°-60°)precipitated a gum which contained the MM 4550 (Complex).

The gum was collected by centrifugation, redissolved in 50 ml ofdichloromethane and extracted with 50 ml of water containing 1 g bariumiodide and 1 g barium carbonate by shaking for 2 minutes. The solidspresent were filtered off and the two phases separated. The aqueousphase containing the MM 4550 (Complex) as the barium salt was adjustedto pH 6.5 and freeze dried to yield 317 mg of a crude preparation of MM4550 (Complex) with an I₅₀ of 0.001 μg/ml.

DESCRIPTION 7 Preparation of Partially Purified Antibiotic Complexcontaining MM 4550A and MM 13902 using Tetra n-butyl Ammonium HydrogenSulphate

A crude preparation of MM 4550 (Complex) prepared as in was redissolvedin water (12.5 g in 125 ml) and extracted with 125 ml of 10% w/v tetran-butyl ammonium hydrogen sulphate in dichloromethane. The two phaseswere separated and the dichloromethane back extracted with 100 ml waterat 2° C. containing 190 mg sodium iodide by stirring slowly andadjusting the aqueous phase to pH 6.4 with 2% NaHCO₃. The solution wasfreeze dried and the dry solid washed with acetone to yield 88 mg of apreparation mixed sodium salts of MM 4550A and MM 13902 having an I₅₀ of0.0002 μg against Escherichia coli β-lactamase. The recovery ofantibiotics was 70%.

The antibacterial activity of mixtures of ampicillin and materialprepared as described in Procedure 7 was determined by the serialdilution method in nutrient agar. The minimum inhibitory concentrationswhich were obtained are given in Table 2.

                                      TABLE 2                                     __________________________________________________________________________    Synergism Between Ampicillin And MM 4550 (Complex)                                               Ampicillin + MM                                                         Ampicillin                                                                          4550 (Complex) at                                          Organism     Alone 0.1 μg/ml                                                                        1 μg/ml                                                                         10 μg/ml                                     __________________________________________________________________________    E. coli B11  >500  >500  500  50                                              E. coli JT417                                                                              250   250   250  50                                              E. coli JT39 >500  >500  500   25*                                            Shigella sonnei S239                                                                       125   125   125   25*                                            Klebsiella aerogenes A                                                                     125   25     5*  <0.1*                                           Klebsiella aerogenes                                                          IP282        50    50    12.5  0.5*                                           Proteus mirabilis 889                                                                      >500  >500   50  0.5                                             Proteus mirabilis 247                                                                      >500  125    5.0 0.5                                             Proteus morganii F                                                                         50    50     25  0.5                                             Proteus rettgeri R110                                                                      250   125   25*   0.25*                                          Staph. aureus (Russell)                                                                    250   125   <0.1  0.25                                           Staph. aureus (Russell H)                                                                  >500  <0.1  <0.1 <0.1                                            __________________________________________________________________________     *Partial inhibition by MM 4550 (Complex) alone at these concentrations.       Similar synergistic effects were observed for mixtures of Amoxycillin and     MM 4550 (Complex).                                                       

DESCRIPTION 8 Preparation of Partially Purified Antibiotic Complexcontaining MM 4550A and MM 13902 using cetyldimethylbenzylammoniumchloride

Freeze dried product (I₅₀ =0.02 μg/ml) from a Farnell carbon columneluted with acetone/water as in Procedure 4 was dissolved in distilledwater at a concentration of 13 mg/ml. The solution was adjusted to pH6.5.

100 ml of the solution was extracted with an equal volume of 0.1%cetyldimethylbenzylammonium chloride in dichloromethane. The phases wereseparated by centrifugation and the organic phase back extracted with100 ml of 0.05% sodium iodide solution. The phases were separated andany dichloromethane in the aqueous phase removed by maintaining thesolution under reduced pressure for 10 minutes.

The aqueous solution was freeze dried and the product of freeze dryingwashed three times with 50 ml portions of acetone. The acetone washedproduct was dried in a vacuum desiccator to yield a light brown powder(4.3 mg; I₅₀ =0.002 μg/ml).

DESCRIPTION 9 Preparation of Partially Purified Antibiotic Complexcontaining MM 4550A and MM 13902 using Gel Filtration

1 g of crude MM 4550 (Complex), I₅₀ 0.2 μg/ml, prepared by the method ofDescription 4 was chromatographed on Sephadex G25 (fine grade), usingacetone/water 4:6 v/v as eluant. The column dimensions were 2.5 cm×32 cmand elution was carried out at 1.5 ml/min. The active fractions werecombined, concentrated in vacuo below 30° C. and freeze dried to yield22 mg amorphous buff coloured solid having an I₅₀ of 0.005 μg/ml. Therecovery was 88% and purification 40-fold.

DESCRIPTION 10 Preparation of Purified Antibiotic Complex containing MM4550A and MM 13902 using Cellulose Chromatography

MM 4550 (Complex) (610 mg) prepared as in Procedure 7 waschromatographed on a 2.5 cm×32 cm column of cellulose (Whatman CC 31)and eluted with a mixture of isopropanol/water, 7/3 (v/v) at 1.5 ml/min.The antibacterially active fractions were combined, concentrated invacuo, below 30° C. and freeze dried to yield 40 mg of brown amorphoussolid preparation of MM 4550 (Complex) I₅₀ of 0.00007 μg/ml. The verysmall value of the I₅₀ indicates that the active material is of improvedpurity.

On a different occasion the above procedure yielded a material having anI₅₀ of 0.001 μg/ml. This material had the antibacterial activity shownin Table 3 when determined by a standard microtitre method in Oxoidsensitivity broth using light inocula (1% of overnight broth).

                  TABLE 3                                                         ______________________________________                                        Antibacterial Activity Of Mixture Of MM 4550A And MM 13902                    Having An I.sub.50 Of 0.001 μg/ml                                          ______________________________________                                                               MIC                                                    Organism               in μg/ml                                            ______________________________________                                        Staphylococcus aureus (Oxford)                                                                       7.5                                                    Staphylococcus aureus (Russell)                                                                      7.5                                                    Streptococcus faecalis 125                                                    Bacillus subtilis      0.9                                                    Escherichia coli (10418                                                                              3.7                                                    Escherichia coli (B11) 15                                                     Klebsiella aerogenes   1.8                                                    Enterobacter cloacae   62                                                     Proteus mirabilis      7.5                                                    Providentia stuartii   7.5                                                    Acinetobacter anitratus                                                                              0.9                                                    Pseudomonas aeruginosa 250                                                    Serratia marcescens    7.5                                                    Salmonella typhimurium 15                                                     Shigella sonnei        7.5                                                    ______________________________________                                    

DESCRIPTION 11 MM 4550A

MM 4550A may be recognised by its properties which are as follows: MM4550A is an acidic solid which in the form of a sodium salt has thefollowing characteristics:

i. It is highly soluble in water, soluble in methanol and substantiallyinsoluble in chloroform, diethylether and hydrocarbons.

ii. In aqueous solution, it has a characteristic ultra-violet spectrumwith absorption maxima at about 238 nm and at about 287 nm.

iii. When present at 0.4% w/w in a freshly prepared KBr disc, it has acharacteristic infra-red spectrum which has absorption maxima at interalia about 3450, 2950, 1765, 1695, 1510, 1390 and 1260 cm⁻¹. If afurther spectrum is taken about one week after the preparation of theKBr. disc, the spectrum shows considerable changes, for example, thelarge peak previously at about 1765 cm⁻¹ is considerably reduced in sizeor is absent.

iv. It has a characteristic n.m.r. spectrum when taken in D₂ O whichspectrum possess inter alia (a) a pair of low field doublets centeredapproximately at 2.45ρ and 3.65ρ with coupling constants ofapproximately 15 Hz; (b) a doublet centered at approximately 8.55ρ and(c) a sharp singlet at approximately 7.95ρ.

v. When run over cellulose in a thin layer chromatography system, it hasthe following very approximate R_(f) values:

(a) butanol/isopropanol/water--7:7:6 v/v; R_(f) =0.7

(b) isopropanol/water--7:3 v/v; R_(f) =0.6

(c) n-butanol/ethanol/water--7:7:6 v/v; R_(f) =0.7

(d) n-propanol/water--4:1 v/v; R_(f) =0.6

vi. It possesses antibacterial activity against various speciesincluding inter alia, strains of Staphylococcus aureus, Bacillussubtills, Escherichia coli, Klebsiella aerogenes, Proteus mirabilis,Acinetobacter anitratus, Serratia marcescens and Shigella sonnei.

vii. It possesses enzyme inhibitory activity against the β-lactamasesproduced by various species including, inter alia, Escherichia coli,Klebsiella aerogenes and Staphylococcus aureus. It has an I₅₀ (ashereinafter defined) of less than 0.0001 μg/ml against the β-lactamaseof Escherichia coli B11

viii. When mixed with ampicillin, it synergyses its activity againstorganisms including strains of Escherichia coli, Klebsiella aerogenes,Proteus mirabilis, Proteus morganii and Staphylococcus aureus Russell.

ix. Amino acid analysis indicates that the material is not a polypeptide or protein. No α-aminoadipic acid is found after acidhydrolysis.

x. It reacts with Ehrlich's reagent (300 mg of4-dimethylaminobenzaldehyde dissolved in a mixture of 54 ml n-butanol, 9ml ethanol and 9 ml concentrated hydrochloric acid) to produce a bluecolour on paper chromatograms and tlc sheets.

ix. It is not a general enzyme poison and does not inhibit the followingenzymes at concentrations in excess of those required to inhibit theβ-lactamase of Escherichi coli: monoamine oxidase, carbonic anhydrase,dopa decarboxylase, trypsin, chymotrypsin or urease.

EXAMPLE 1 Preparation of MM 4550 (Complex) and Separation into MM 4550Aand MM 13902

The isolation procedures detailed in the Description section may beutilized in a variety of sequences. A particularly suitable sequence iscarbon adsorption, ion pair extraction and cellulose chromatography asdescribed below:

Culture filtrate (340 l) (obtained as in Description 3) was percolatedby upward flow at 800-1200 ml/min. through a column (9" diameter×21"high) packed with Farnell B0 carbon. The carbon had been used before foradsorption of MM 4550 (Complex) and was regenerated by washing with thefollowing reagents:

0.5 N NaOH; 0.2 N NaOH/acetone (3:2); water; N HCl; water; phosphatebuffer pH 6; water.

The column was washed with water (20 l) to displace the culture filtrateand eluted by downward flow with acetone/water 2:8 v/v at 200-250ml/min. One liter fractions were collected. Fractions containing the MM4550 (Complex) as determined by an antibacterial diffusion assay usingKlebsiella aerogenes were combined (11 l) and concentrated byevaporation in vacuo below 30° C. to 5.5 l. The recovery of MM 4550(Complex) at this stage was 20%.

The concentrate was cooled to 2° C., 5.5 l of 2% w/v tetran-butylammonium hydrogen sulphate in dichloromethane at -5° C. was addedand the two phases mixed together for 2 minutes. The dichloromethanephase was separated and added to one liter of sodium iodide solution(0.6%) at 0° C. The two phases were stirred gently and the aqueous phasegradually adjusted to between pH 6.5-7.0 with 2% aqueous solution ofNaHCO₃. The aqueous phase was separated and freeze dried. The driedsolid was extracted with dry acetone to dissolve out excess sodiumiodide and the acetone removed from the insoluble residue in vacuo, toyield a buff coloured powder (1.1 g). Overall recovery of MM 4550(Complex) at this stage was 10%. The purification calculated on totaldissolved solids in the culture filtrate was 125-fold.

A 2.5 cm diameter column was packed with cellulose powder (Whatman CC31) in isopropanol/water, 7:3 v/v, to a height of 32 cm. 500 mg of thebuff powder was dissolved in 2 ml isopropanol/water 7:3 v/v andchromatographed using the same solvent at a flow rate of 1.5 ml/min.Fractions (3 ml) were collected from the column and those showing UVabsorption maxima at about 285 nm were combined, concentrated and freezedried to yield NM 4550 (Complex). Previous experiments have establishedthat fractions absorbing at about 285 nm contained enzyme inhibiting,antibacterially active material.

The yield of freeze dried MM 4550 (Complex) was 35 mg. It had a brownamorphous appearance and an I₅₀ of 0.0001 μg/ml. The recovery of activematerial at this stage was 3% overall and the purification 600-foldoverall.

The antibacterial activity of this preparation was determined by thestandard Microtitre method in Oxoid sensitivity test broth using lightinocula. The resulting minimum inhibitory concentrations were similar tothose shown in Table 3 hereinbefore.

Analysis of the preparation of thin layer chromatography on cellulose(Eastman Kodak "Chromagram" sheets) developed with n-butanol;isopropanol:water (7:7:6) separated two active substances, one with anapproximate R_(f) of 0.54 which was designated MM 4550A and one with anapproximate R_(f) of 0.72 which was designated MM 13902. Both thesematerials may be detected by bioautography on a variety of organismsincluding B. subtilis, Staphylococcus aureus (Oxford), Staphylococcusaureus (penicillin resistant), Escherichia coli (penicillin sensitiveand resistant), Salmonella typhimurium, Proteus mirabilis, Proteusmorganii and Klebsiella aerogenes. Some of the results obtained aresummarised in Table 4.

In a further experiment, the two substances were separated on acellulose thin layer developed with isopropanol:water (8:2) andextracted from the cellulose with phosphate buffer. Each extract wasassayed for β-lactamase inhibition and both substances were shown to beinhibitors of Escherichia coli β-lactamase. Both substances were alsoshown to be synergistic with benzylpenicillin against Klebsiellaaerogenes.

                  TABLE 4                                                         ______________________________________                                        Antibacterial Activity Of MM 4550A And MM 13902 As                            Determined By Bioautography (TLC With Cellulose And                           Butanol/Isopropyl Alcohol/Water 7:7:6 v/v)                                                     Zone Diameter                                                                 on Bioautography                                                                MM 4550A   MM 13902                                                           zone in    zone in                                         Organism           mm at R.sub.f 0.58                                                                       mm at R.sub.f 0.70                              ______________________________________                                        Klebsiella aerogenes                                                                             20.0 mm    20.0 mm                                         Proteus mirabilis  17.5 mm    13.5 mm                                         Proteus morganii   16.0 mm    9.5 mm                                          Salmonella typhi   19.5 mm    21.0 mm                                         Escherchia coli 10418                                                                            20.0 mm    13.5 mm                                         Escherchia coli B11                                                                              17.5 mm    10.5 mm                                         Enterobacter aerogenes                                                                           6.5 mm     No Zone                                         Staphylococcus aureus (Oxford)                                                                   13.0 mm    4.0 mm                                          Staphylococcus aureus (Russell)                                                                  12.0 mm    4.0 mm                                          Bacillus subtilis  24.0 mm    15.0 mm                                         ______________________________________                                    

EXAMPLE 2 Preparation of MM 4550 (Complex) and Separation into MM 4550Aand MM 13902

Culture filtrate (1100 l) from the fermentation of Streptomycesolivaceua ATCC 31126 at pH 6.5 and 5° C. were percolated at 3.2 l/min.through a column (0.3 m×1 m) packed with Darco granular carbon. [Thecarbon had already been used for adsorption of MM 4550 (Complex) and wasregenerated before use by washing with the following reagents:

0.5 N NaOH; 0.2 N NaOH/acetone (3:2); water; N HCl; water; phosphatebuffer, pH 6; water].

The column was washed with water (60 l) to displace the culture filtrateand eluted with acetone/water (2:8 v/v) at 30° C. at 1.1 l/min. 60 l wascollected followed by 5×10 l fractions. Those fractions containing theMM 4550 (Complex), as determined by the antibacterial diffusion assayusing Klebsiella aerogenes were pooled (40 l) and concentrated in vacuobelow 30° C. to 32 l. The recovery of MM 4550 (Complex) at this stagewas 15%.

The concentrate was cooled to 5° C., 16 l of 0.2%cetyldimethylbenzylammonium chloride in dichloromethane at 5° C. wasadded and the two phases mixed for 5 mins. The dichloromethane phase wasseparated and added to 3.75 l sodium iodide solution (0.4%) at 5° C. Thetwo phases were mixed for 5 mins., the aqueous phase was separated andfreeze dried. The dry solid was extracted with dry acetone to dissolveout excess sodium iodide and the residual solid was dried in vacuo toyield a buff coloured powder (2.87 g). Overall recovery of MM 4550(Complex) at this stage was 6%. The purification calculated on totaldissolved solids in the culture filtrate was 160-fold. A 63 mm diametercolumn was packed with cellulose powder (Whatman CC 31) inisopropanol-water (7:3 v/v) to a height of 300 mm. 2.0 g of the buffcoloured powder was dissolved in 5 ml isopropanol/water (7:3 v/v) andchromatographed in the same solvent at 3 ml/min. Fractions containing MM4550 (Complex), as determined by assay against Klebsiella aerogenes werepooled, concentrated by evaporation in vacuo below 30° C. and freezedried to yield a brown solid (207 mg.). The recovery of MM 4550(Complex) for this stage was 51% and the purification was 5-fold. A 16mm diameter column was packed with cellulose powder (Whatman CC 31) inn-propanol/water (4:1 v/v) to a height of 300 mm. 198 mg of the brownsolid was dissolved in water/n-propanol (1:1) and chromatographed inn-propanol/water (4:1 v/v) at a flow rate of 0.5 ml/min. Fractions (6ml) were collected, bioassayed against Klebsiella aerogenes and the UVspectrum of each fraction measured. Fractions 23-28, having a UV maximumat about 305 nm, contained the di-sodium salt of MM 13902. Thesefractions were pooled, concentrated under vacuum and freeze dried togive 30 mg of solid. This preparation showed only one zone of antibioticactivity at R_(f) 0.77 on thin layer chromatography using Eastman Kodakcellulose plates with n-propanol/water (4:1 v/v). The zone was detectedby bioautography on Bacillus subtilis. (Fractions 29-40, having a UVabsorption maximum at about 285 nm, contained MM 4550A. They werecombined, concentrated under vacuum and freeze dried to give 53 mg ofsolid which contained a salt of MM 4550A contaminated with a smallquantity of a salt of MM 13902).

DESCRIPTION 12 Organisms

The property of producing MM 4550 (Complex) was first discovered in thecultures ATCC 21379, ATCC 21380, ATCC 21381 and ATCC 21382 which hadbeen isolated from soil samples obtained from Spain, New Zealand, SouthAfrica and Israel respectively. In the laboratory, these culturesappeared identical and all were identified as Streptomyces olivaceus, byDr. Bousfield, the actinomycete expert at the National Collection ofIndustrial Bacteria (NCIB), Torry Research Station, Aberdeen, Scotland,using the widely accepted 1967 classification of Hutter [Systematic derStreptomyceten, S. Karger, Basel, 382 pages]. MM 4550A production hassince been found in other streptomyces species as may be seen from Table4.

S. olivaceus was first described by Waksman in 1919 [Cultural Studies ofSpecies of Actinomyces, Soil. Sci., 8, 71-215]. Subsequently, in 1960, agroup of Russian workers described a species with very similarcharacteristics but which they named Actinomyces (Streptomyces)fulvoviridis. [Kuchaeva, et al., Trud. Inst. Mikrobiol., Akad Nauk.SSSR., 8, 226-253 (1960)].

Micro-organisms of the genus Streptomyces are extremely variable intheir morphological and physiological characteristics depending on theconditions under which they are grown. Descriptions of many species hadbeen published before the existence of this extreme variability had beenrecognised, with consequent duplication and synonymy. Hutter (1967)lists 25 species as being synonymous with S. olivaceus including S.fulvoviridis. To resolve the confusion in nomenclature andclassification, the International Streptomyces Project was begun in1962, Shirling, et al., Int. J. Syst. Bacteriol, 18, 69-189 (1968).Collaborators in this Project have carried out a series of studies aimedat producing accurate descriptions of species of Streptomyces understandard conditions. In this scheme, standard descriptions have beenproduced of type strains of some 400 named species includingStreptomyces olivaceus and Streptomyces fulvoviridis.

The I.S.P. description of S. olivaceus and S. fulvoviridis differ in twocharacters only: (i) the form of the sporophore and (ii) ability toutilise inositol as sole carbon source.

S. olivaceus is described as follows:

Spore chain morphology: Section Spirales, with open spirals intergradingthrough flexuous spore chains suggestive of Section Rectiflexibiles.Mature spore chains generally long, often with more than 50 spores perchain.

Carbon utilization: D-Glucose, L-arabinose, D-xylose, i-inositol,D-mannitol, D-fructose and rhamnose are utilised for growth. No growthor only trace of growth on sucrose and raffinose.

S. fulvoviridis is described as follows:

Spore chain morphology: Section Rectiflexibiles. Mature spore chainsmoderately short with 10-50 spores per chain. Carbon utilisation:D-Glucose, L-arabinose, D-xylose, D-mannitol, D-fructose and rhamnoseare utilised for growth; utilisation of sucrose is doubtful. No growthor only trace of growth on i-inositol or raffinose.

Characterisation of a number of cultures named or synonymous with S.olivaceus or S. fulvoviridis and other related species was determinedusing the standard methods and media (Shirling, et al., Int. J. Syst.Bacteriol. 16, 313 340, (1966) recommended in the InternationalStreptomyces Project (ISP).

The cultures were derived from different sources. ATCC 21379, 21380,31381, 21382 were isolated from soil on the basis of producing MM 4550(Complex). The other strains were obtained from various culturecollections for comparison purposes.

Results of the tests for production of MM 4550 (Complex), colour ofaerial mycelium, sporophore shape, colour of substrate mycelium solublepigment production, melanin production and carbon source utilization aregiven in Tables 4-8. For all strains the spore surface, as seen byelectron microscopy, is smooth.

From the ISP description it is clear that the spiral growth of thesporophore in S. olivaceus is a variable character. True spirals wereobserved with a variable frequency in the type species of S. olivaceus,i.e. ATCC 3335. The majority of the sporophores were long. No truespirals were observed from the cultures ATCC 21379, 21380, 21381 or21382 although the sporophores were long and showed a tendency to spiralamong a background of Rectiflexibiles types. However, in two S.olivaceus strains, NCIB 8238 and NCIB 8509, the majority of sporophoreswere of the Rectiflexibiles type. In NCIB 8238 they were of mediumlength while those of NCIB 8509 were long.

The sporophores observed from ATCC 15863, the type species of S.fulvoviridis, were shorter than those seen from isolates ATCC 21379,21380, 21381 and 21382 and were only of the Rectiflexibiles types. Inrespect of this character therefore, the isolates ATCC 21379, 21380,21381 and 21382 correspond fairly well but not exactly with the S.olivaceus description.

Isolates ATCC 21379, 21380, 21381 and 21382 show some variability withregard to inositol utilization which is the other differentiatingcharacter according to the I.S.P. type species descriptions between thespecies S. olivaceus and S. fulvoviridis (Table 8). ATC 31126 and ATCC21380 utilize inositol, which is characteristic of S. olivaceus, whilethe other strains show doubtful or negative utilization. However, it isnot generally considered satisfactory to separate off a species on thebasis of the ability to utilize a single carbohydrate. Such a differencemight arise by a single gene mutation and is often considered to be ofstrain significance only. The difference in sugar utilization by the S.olivaceus strains NCIB 8138, NCIB 8509, ATCC 21549, ATCC 12019 and ATCC3335 suggest that many authorities do not consider them of speciessignificance. Thus it is likely that ATCC 21379, 21380, 21381 and 21382are properly designated S. olivaceus.

Unless further studies uncover more important differences between thosecultures presently named S. olivaceus and S. fulvoviridis, it ispossible that they will eventually be internationally recognized as asingle species. In this case the correct name will be S. olivaceus, andthis will also include those cultures listed by Hutter as synonymouswith S. olivaceus.

Examination of culture filtrates of the strains of S. olivaceus andrelated species for the production of MM 4550 (Complex) may be carriedout as follows:

Culture filtrates of the listed strains were spotted on Whatman No. 1paper strips at 20 μl per origin and chromatographed inn-butanol/isopropanol/water (7:7:6) overnight in the cold. Another setof strips were run in n-butanol/glacial acetic acid/water (12:3:5) alsoin the cold overnight. A partially purified sample of MM 4550 (Complex)was also run in the two systems at the same time as a market.

Alternatively, it is possible to extract 25 ml of clarified brew with12.5 ml of 0.2% cetyllunzylammonium chloride in dichloromethane,separate the phases, retain the organic phase, add 2.5 ml of sodiumiodide solution (0.5%), shake, separate the phases, retain the aqueousphase and use this chromatographically, for example, by spotting 5μ ontothin layer chromotography strips and run in n-butanol/isopropanol/water,7:7:6.

                  TABLE 5                                                         ______________________________________                                        Ability Of Cultures Streptomyces Olivaceus,                                   Streptomyces fulvoviridis; And Related                                        Species To Produce MM 4550 (Complex)                                                                MM 4550 (complex)                                       Culture               Production                                              ______________________________________                                        Streptocmyces olivaceus                                                                      ATCC 21379 +                                                   Streptomyces olivaceus                                                                       ATCC 31126 +                                                   Streptomyces olivaceus                                                                       ATCC 21380 +                                                   Streptomyces olivaceus                                                                       ATCC 21381 +                                                   Streptomyces olivaceus                                                                       ATCC 21382 +                                                   Streptomyces olivaceus                                                                       NCIB 8238  +                                                   Streptomyces olivaceus                                                                       NCIB 8509  +                                                   Streptomyces flavovirens                                                                     ATCC 3320  +                                                   Streptomyces flavus                                                                          ATCC 3369  +                                                   Streptomyces fulvoviridis                                                                    ATCC 15863 +                                                   Streptomyces argenteolus                                                                     ATCC 11009 +                                                   Streptomyces sioyaerisis                                                                     ATCC 13989 +                                                   ______________________________________                                         (Streptomyces olivaceus ATCC 21549, ATCC 12019 and ATCC 3335 did not          produce MM 4550 (complex); ATCC 15863 has also been deposited as RIA 660)

                                      TABLE 6                                     __________________________________________________________________________    Streptomyces Olivaceus, Streptomyces Fulvoviridis And Related Species         Colour And Morphology Of Mature Sporulating Aerial Mycelium                   On ISP Media After 14 Days Growth                                                                                Sporophore                                                                    Morphology                                 Culture                                                                              SS     YM     GA     OM     mid size                                   __________________________________________________________________________    ATCC 21379                                                                           Grey   Grey   Grey/White                                                                           Grey   Long RF                                    ATCC 31126                                                                           Grey/Brown                                                                           Grey/Brown                                                                           Grey   Grey   Long RF                                    ATCC 21380                                                                           Grey   Grey   Grey   Grey   Long RF                                    ATCC 21381                                                                           Grey   Grey   Pale Grey                                                                            Grey/White                                                                           Long RF                                    ATCC 21382                                                                           Grey/White                                                                           Grey   Grey   Grey   Long RF                                    NCIB 8238                                                                            Grey/White                                                                           Grey   Grey   Grey   Medium RF                                  NCIB 8509                                                                            Grey   Grey   Grey   Grey   Long RF                                    ATCC 21549                                                                           Grey   Grey   Grey   Grey   Long S                                     ATCC 12019                                                                           Grey   Grey   Grey   Grey   Long RF/S                                  ATCC 3335                                                                            Grey   Grey   Grey   Grey   Long RF/S                                  ATCC 15863                                                                           Grey/White                                                                           Grey   Grey/White                                                                           Grey   Medium RF                                  ATCC 3320                                                                            Grey/blue                                                                            Grey   Grey/Green                                                                           Grey   Long RF                                    ATCC 3369                                                                            Grey   Grey   Grey   Pale grey/                                                                    brown  Medium RF/RA                               ATCC 11009                                                                           Pale Grey                                                                            Grey/brown                                                                           Grey/White                                                                           Grey/Green                                                                           Medium RF/RA                               ATCC 13898                                                                           White/Grey                                                                           White  White/yellow                                                                         Grey/White                                                                           Short S                                    __________________________________________________________________________     SS = Inorganic salts  starch agar (ISP Medium 4)                              YM = Yeast extract  malt extract agar (ISP Medium 2)                          GA = Glycerol  asparagine agar (ISP Medium 5)                                 OM = Oatmeal agar (ISP Medium 3)                                              RF = Rectiflexibiles                                                          RA = Retinaculiaperti                                                         S = Spirales                                                             

                                      TABLE 7                                     __________________________________________________________________________    Streptomyces Olivaceus, Streptomyces Fulvoviridis And Related Species         Colour Of Substrate Mycelium As Viewed From The Reverse Side                  Culture SS      YM      GA      OM                                            __________________________________________________________________________    ATCC 21379                                                                            Olive   olive brown                                                                           grey/brown                                                                            olive green                                   ATCC/31126                                                                            olive brown                                                                           olive brown                                                                           brown   olive brown                                   ATCC 21380                                                                            olive   olive green                                                                           olive brown                                                                           olive yellow                                  ATCC 21381                                                                            dark brown                                                                            dark brown                                                                            olive green                                                                           olive green                                   ATCC 21382                                                                            olive   olive   brown   olive green                                   NCIB 8238                                                                             brown   olive/brown                                                                           brown   olive yellow                                  NCIB 8509                                                                             brown   olive   olive brown                                                                           olive yellow                                  ATCC 21549                                                                            buff/black                                                                            brown/black                                                                           brown/black                                                                           buff/grey                                     ATCC 12019                                                                            buff    buff    buff    buff                                          ATCC 3335                                                                             brown   brown   brown   grey                                          ATCC 15863                                                                            brown/grey                                                                            dark brown                                                                            olive brown                                                                           olive yellow                                  ATCC 3320                                                                             yellow/brown                                                                          olive/brown                                                                           olive/brown                                                                           orange/brown                                  ATCC 3369                                                                             buff/grey                                                                             yellow brown                                                                          buff/green                                                                            yellow                                        ATCC 11009                                                                            black   black/yellow                                                                          black/yellow                                                                          grey/green                                    ATCC 13989                                                                            Orange  yellow  yellow  colourless                                    __________________________________________________________________________

                  TABLE 8                                                         ______________________________________                                        Streptomyces Olivaceus, Streptomyces Fulvoviridis And                         Related Species - Production Of Pigments In Culture Media                     ______________________________________                                               Soluble Non-Melanoid Pigments                                                                     *Melanin                                           Culture  SS      YM      GA      OM    Production                             ______________________________________                                        ATCC 21379                                                                             -       -       -       +     -                                                                       slight                                                                        yellow                                       ATCC 31126                                                                             -       -       -       -     -                                      ATCC 21380                                                                             -       -       -       -     -                                      ATCC 21381                                                                             -       -       -       -     -                                      ATCC 21382                                                                             -       -       -       -     -                                      NCIB 8238                                                                              +       -       +       +     -                                               slight          slight  slight                                                brown           brown   brown                                        NCIB 8509                                                                              -       -       -       +     -                                                                       slight                                                                        yellow                                       ATCC 21549                                                                             -       +       +       +     -                                                       slight  slight red/                                                                           slight                                                        brown   brown   brown                                        ATCC 12019                                                                             -       -       -       -     -                                      ATCC 3335                                                                              -       -       -       -     -                                      ATCC 15863                                                                             -       -       -       -     -                                      ATCC 3320                                                                              +       -       -       -     -                                               slight                                                                        pink                                                                 ATCC 3369                                                                              -       -       -       -     -                                      ATCC 11009                                                                             -       +       -       -     -                                                       slight                                                                        brown                                                        ATCC 13989                                                                             -       +       -       -     -                                                       slight                                                                        orange                                                       ______________________________________                                         + = Pigment produced                                                          - = Pigment not produced                                                      *Tested on peptoneyeast-iron agar (ISP6), tyrosine agar (ISP17) and           tryptoneyeast broth (ISP 1)                                              

                                      TABLE 9                                     __________________________________________________________________________    Streptomyces Olivaceus, Streptomyces Fulvoviridis And Related Species         Carbon Utilization Tests                                                      Culture                                                                              Rhamnose                                                                            Raffinose                                                                          Sucrose                                                                            Inositol                                                                           Glucose                                                                            Xylose                                                                            Fructose                                                                           Marmitol                                                                           Arabinose                      __________________________________________________________________________    ATCC 21379                                                                           +     -    -    ± +    +   +    +    +                              ATCC 31126                                                                           +     -    -    +    +    +   +    +    +                              ATCC 21380                                                                           +     -    -    +    +    +   +    +    +                              ATCC 21381                                                                           +     -    -    -    +    +   ± ± +                              ATCC 21382                                                                           +     +    -    -    +    +   +    +    +                              NCIB 8238                                                                            +     -    -    -    +    +   +    +    +                              NCIB 8509                                                                            +     ± -    -    +    +   +    ± +                              ATCC 21549                                                                           -     -    -    +    +    +   +    +    +                              ATCC 12019                                                                           +     ± +    +    +    +   +    +    +                              ATCC 3335                                                                            +     -    ± +    +    +   +    +    +                              ATCC 15863                                                                           +     -    -    -    +    +   +    +    +                              ATCC 3320                                                                            +     -    ± -    +    +   +    +    +                              ATCC 3369                                                                            +     -    ± ± +    +   +    +    +                              ATCC 11009                                                                           +     -    -    +    +    +   +    +    +                              ATCC 13989                                                                           -     +    +    +    +    +   +    +    -                              __________________________________________________________________________     + denotes the compound is utilized                                            - denotes the compound is not utilized                                        ± denotes utilization is doubtful or very poor                        

EXAMPLE 3 A Preferred Isolation Procedure for the Preparation of MM13902

A stock of spores of S. olivaceus ATCC 31126 was maintained by storagein tubes of dry soil in a closed container with a dessicant at atemperature of 20° C. A small quantity of soil stock (approximately 20mg) was transferred aseptically to a 500 ml Erlenmeyer flask containing100 ml of the following medium:

    ______________________________________                                        Constituent        Amount (g/liter)                                           ______________________________________                                        Glucose monohydrate                                                                              20.0                                                       Soya-bean flour    10.0                                                       Deionised water to 1 liter                                                    ______________________________________                                    

The pH was adjusted to 6.5 before sterilisation. The soya bean flour was`Arkasoy 50` as supplied by British Arkady Co. Ltd., Old Strafford,Manchester, England.

The flask was incubated on a rotary shaker (240 r.p.m.) for about 30hours at 28° C. 2 ml of the resulting vegetative growth was then used toinoculate a solid agar slant in a Roux bottle. The agar medium had thefollowing compositions:

    ______________________________________                                        Constituent        Amount                                                     ______________________________________                                        V-8 vegetable juice                                                                              20.0 ml                                                    Agar               20.0 g                                                     Deionised water to 1 liter                                                    ______________________________________                                    

The pH was adjusted to 6.0 before sterilization. (V-8 juice isobtainable from Campbell's Soups Ltd., Kings Lynn, Norfolk, England).The inoculation was spread on the agar surface by rocking the bottlewhich was then incubated upright at 30° C. After two days incubation,surplus liquid in the bottle was removed by pipette and incubationcontinued for a further 4 days.

It had been previously found that the development of actinophage in theslant culture is suppressed if this method of preparation of the Rouxbottle culture is adopted.

50 ml of sterilised deionised water containing 0.02% Tween 80 was addedto a Roux bottle culture and the spores suspended by shaking. This sporesuspension was then added as inoculum to 75 liters of sterilised seedstage medium in a 100 liter stainless steel fermenter. The compositionof the seed stage medium was as follows.

    ______________________________________                                        Constituent             Amount (g/l)                                          ______________________________________                                        Soya-bean flour (`Arkasoy 50`)                                                                        10.0                                                  Glucose monohydrate     20.0                                                  Tap water to            1 liter                                               ______________________________________                                    

To control foaming, 50 ml of 10% v/v `Pluronic L81` in soyal-bean oilwas added to the fermentation medium before sterilization.

The medium was steam sterilized in the fermenter for 20 minutes at 120°C. The seed stage culture was stirred at 140 r.p.m. with a 7.5 inchdiameter vaned disc agitator and supplied with 75 liters/min sterile airthrough an open ended sparger.

The temperature was controlled at 28° C. and after incubation underthese conditions for 48 hours the contents of the vessel were added asinoculum to 1500 liters of sterile fermentation medium in a 2000 literstainless steel fully baffled fermenter. The fermentation medium had thefollowing composition:

    ______________________________________                                        Constituent            Amount (g/liter)                                       ______________________________________                                        Soya-bean flour (`Arkasoy 50`)                                                                       10.0                                                   Glucose monohydrate    20.0                                                   Chalk (precipitated calcium carbonate)                                                                0.2                                                   Cobalt chloride (COCl.sub.2 . 6H.sub.2 O)                                                              0.001                                                Sodium sulphate (anhydrous)                                                                           1.0                                                   Tap Water to           1500 liters                                            ______________________________________                                    

The pH was adjusted to 6.0 with sodium hydroxide before sterilization. 3liters of 10% `Pluronic L81` in soya bean oil was added beforesterilization to prevent foaming. After sterilization the pH was againadjusted, to 7.0, with sterile sodium hydroxide solution. Thefermentation was stirred at 106 r.p.m., the stirrer shaft being fittedwith two 19 inch diameter vaned disc impellers. Temperatures wascontrolled at 30° C. and airflow at 1200 liters/min. The fermentationwas harvested after 60 hours and clarified by centrifugation.

The material produced above ws arbitrarily assigned an activity of 340l/ml. Assays were determined as described in Description 3.

Culture filtrate (1050 l; 340 units/ml.) at 10° C. and pH8 was extractedwith dichloromethane (310 l) at 10° C. containing cetyldimethylammoniumchloride (1200 g) by pumping the two liquids at predetermined flow ratesthrough an in-line mixer. The phases were separated in a Sharplescontinuous centrifuge having been admixed for about two minutes. Thedichloromethane phase (300 l) was back extracted with aqueous sodiumiodide. The back extraction was performed in four batches using a totalof 7 l water containing 210 g sodium iodide. The phases were separatedby gravity. The aqueous phase was adjusted from pH 7.7 to pH 7.0 withhydrochloric acid and filtered. The sodium iodide extract (7 l)contained 21,900 units/ml.

An ion exchange column was prepared by packing QAE Sephadex A25(obtained from Pharmacia Ltd.) in pH7 phosphate buffer (0.05 M)containing sodium chloride (0.3 M) into a 10 cm diameter glass column toa height of 40 cm. The sodium iodide extract (7 l) at 5° C. waspercolated through the QAE Sephadex at 50 ml/min. The column was elutedwith 0.7 M NaCl in 0.05 M phosphate buffer, pH 7 also at 5° C. at a flowrate of 25 ml/min. 2 l eluate was discarded and 90 fractions (100 ml)were collected. The fractions were scanned in a u.v. spectrophotometerand those fractions showing an absorption maximum at about 305 nm werepooled and adjusted to pH 7 (fractions 50-62, pooled volume 1440 ml71,000 units/ml. It had previously been shown that fractions having amaximum in this region contained MM 13902.

Sodium chloride (5 g/100 ml) was added to the pooled fractions whichwere then percolated at 5° C. through a 6.3 cm diameter column packedwith Amberlite XAD 4 resin (as supplied by Rohm & Haas Ltd.) to a heightof 30 cm at a flow rate of 20 ml/min. The antibiotics were adsorbed tothe resin under these conditions whereas the inorganic impurities werenot. The antibiotics were eluted at room temperature with distilledwater (200 ml) followed by 50% aqueous methanol. The eluate (1 l) wasevaporated below 30° C. under reduced pressure to 70 ml, adjusted to pH7 and freeze dried to a brown solid (1.62 g) which was a partiallypurified salt of MM 13902 with an activity of 37,000 units/mg.

Partially purified MM 13902 di-sodium salt (1.0 g, 37,000 units/mg) waschromatographed on a cellulose column (3.8 cm×30 cm; Whatman CC31cellose) eluted with n-propanol/water (4:1 v/v) at a flow rate of 2.5ml/min. 170 ml eluate was discarded and 100×15 ml fractions collected.The fractions containing MM 13902 di-sodium salt (Nos. 37-43) asdetermined by uv absorption were pooled (89 ml), evaporated below 30° C.under reduced pressure to remove n-propanol and freeze dried to yield ayellowish powder (219 mg) with an activity of 73,000 units/mg.

The above material showed a uv absorption maximum at about 308 nm withan E₁ cm^(1%) of about 343. This material had the i.r. and n.m.r.spectra shown in FIGS. 2 and 3 respectively. The antibacterial activityof the material is shown in Table 10. Elemental analysis of thismaterial indicated that it contained inter alia nitrogen, sulphur andsodium probably in the ratio N:S:Na--2:2:2. The positive and negativemaxima of the circular dichroism curves for di-sodium MM 13902 weredetermined on a Cary-61 recording spectropolarimeter at concentrationsof 0.37 mg/ml at a path length of 1 cm; the results were as follows:

    ______________________________________                                        λ (nm)      Δ E/m                                                ______________________________________                                        186                + 67.24 × 10.sup.-4                                  221                -67.24 × 10.sup.-4                                   286                - 3.3  × 10.sup. -4                                  323                - 8.2  × 10.sup. -4                                  ______________________________________                                    

                  TABLE 10                                                        ______________________________________                                        Antibacterial Activity Of The Di-Sodium Salt of MM 4550A                      (Microtiter Method - Heat Inoculum, 1/100 Overnight Broth)                    ______________________________________                                        Organism              MIC (μg/ml)                                          ______________________________________                                        Bacillus subtilis A   25                                                      Staph. aureus Oxford  20                                                      Staph. aureus Russell 20                                                      Strep. faecalis       100                                                     Enterobacter cloacae N1                                                                             100                                                     Kleb. aerogenes A     2.5                                                     Proteus mirabilis 13  10                                                      Proteus vulgaris WO 90                                                                              5                                                       Prov. stuartii        5                                                       Ps. aeruginosa A      >100                                                    Salmonella typhimurium CT 10                                                                        5                                                       Serratia marcescens US 39                                                                           20                                                      Shigella sonnei       5                                                       ______________________________________                                    

What is claimed is:
 1. A process for the preparation of apharmaceutically acceptable di-basic salt of MM 13902 which is at least50% pure wherein MM 13902 is a solid carboxylic acid of the molecularformula C₁₃ H₁₆ O₈ N₂ S₂ which in the form of a substantially puresodium salt has the following characteristics:(a) in aqueous solution,it has a characteristic ultra-violet spectrum with absorption maxima atabout 305 nm and at about 225 nm substantially as shown in FIG. 1; (b)when present at 0.4% w/w in a freshly prepared KBr disc, it has acharacteristic infra-red spectrum which has absorption maxima at interalia about 3450, 2950, 1750, 1620, 1510, 1400 and 1260 cm⁻¹ ; (c) it hasa characteristic N.M.R. spectrum when taken in D₂ O which spectrumpossesses inter alia (i) a pair of low field doublets centered atapproximately 2.85ρ and 4.00ρ with coupling constants of approximately14 Hz; (ii) a doublet centered at approximately 8.55ρ and (iii) a sharpsinglet at approximately 8.00ρ; (d) it possesses antibacterial activityagainst various species including inter alia, strains of Staphylococcusaureus, Bacillus subtilis, Escherichia coli, Klebsiella aerogenes,Proteus mirabilis, Salmonella typhi and Pseudomonas aeruginosa; and (e)when mixed with ampicillin it synergizes its activity against organismsincluding strains of Escherichia coli, Klebsiella aerogenes, Proteusmirabilis, Proteus morganii and Staphylococcus aureus Russell, whichcomprises chromatographically separating from a solution of MM 4550(Complex) which contains MM 13902 and impurities a fraction consistingessentially of a solution of a pharmaceutically acceptable di-basic saltof MM 13902 and thereby obtaining a pharmaceutically acceptable di-basicsalt of MM 13902 of at least 50% purity.
 2. A process according to claim1 which further comprises isolating those fractions having a UVabsorption maximum at about 305 nm which contain the substantially pure,pharmaceutically acceptable di-basic salt of MM 13902 from the solution.3. A process according to claim 2 wherein the solution of MM 4550(Complex) is an aqueous solution containing electrolytes buffered toapproximate neutrality and the di-basic salt is separated using a basicion exchange resin.
 4. A process according to claim 3 wherein the basicion exchange resin is an ion exchange cellulose or an ion exchangecross-linked dextran.
 5. A process according to claim 2 wherein thesolution of the pharmaceutically acceptable salt of MM 13902 contains amixture of water and a water-miscible organic solvent, and the salt isseparated using an inert support material.
 6. A process according toclaim 5 wherein the water-miscible organic solvent is a lower alkanol.7. A process according to claim 5 wherein the water-miscible organicsolvent is isopropanol and the ratio of ispropanol:water is 7:3.
 8. Aprocess according to claim 2 which comprises desalting the fractionscontaining the pharmaceutically acceptable di-basic salt of MM 13902before isolating the salt.
 9. A process according to claim 1 whichcomprises gel filtering the pharmaceutically acceptable di-basic salt ofMM 13902 before isolation.
 10. A process according to claim 2 whereinthe pharmaceutically acceptable di-basic salt of MM 13902 is obtained insolid form by removing the solvent under mild conditions.
 11. A processaccording to claim 10 wherein the solvent is evaporated under reducedpressure and the fractions containing a pharmaceutically acceptabledi-basic salt of MM 13902 are freeze-dried.
 12. A process according toclaim 2 wherein the di-basic salt of MM 13902 is in the form of analkali metal salt.
 13. A process according to claim 2 wherein thedi-basic salt of MM 13902 is in the form of the sodium, potassium,calcium, magnesium, aluminum or ammonium salt.
 14. A process accordingto claim 2 for the preparation of the di-sodium salt wherein thefraction separated consists essentially of a solution of the di-sodiumsalt of MM 13902.